Prof. Dr. Luciano Marraffini, The Rockefeller University

An Interview with the 2015 Hans Sigrist Prize Winner

HSF: How did you get interested in the field of bacteriology originally?

Marraffini: I think that was in college, probably, I have always been interested in molecular biology, to understand the biology of the simplest organisms, to understand bacterial life cycles and what they do. I decided to do my Ph.D. at the University of Chicago, where one class I really liked was bacterial pathogenesis, to learn how all these bacteria were causing disease, and the professor of that class became my Ph.D. supervisor - I chose to do a Ph.D. on microbiology based on that class.

HSF: It is amazing how one professor can have an effect on your life sometimes.

Marraffini: Yes, that's really true.

HSF: Can you explain your specific sub-field of adaptive immunity to our readers who are not experts in your field?

Marraffini: I will do my best. Bacteria, they are a simple organism, if you will. Prof. Endimiani (University of Bern) and I will not describe them as simple, as they are much more complex than people think, but in a way, they are just one cell. But still, they are infected by viruses; these viruses are known as bacteriophages. Viruses of bacteria are extremely potent; they can wipe out a population in a matter of a few hours, if not less. So, bacteria need to develop mechanisms to defend against these viral infections. There are many different mechanisms, many of them are just called resistance mechanisms, where bacteria become resistant to the virus. However, about ten years ago, a system was discovered that resembles an adaptive immune system of bacteria, similar to the adaptive immune system that we have that defends our cells from viral infections.

The way that the viral infection of bacteria works is that the virus will interact with the surface of the bacteria – basically sit on the surface of the bacteria, and then it will inject the genetic material of the virus. The bacteria, through this adaptive immune system, which is called CRISPR, can grab a little piece of the viral DNA and use that, transferring information of the infection to the bacteria and incorporating that viral DNA in its own DNA in the CRISPR locus. The CRISPR locus then becomes a collection of little snippets of viral information that are a history of infection of the bacterium. The bacterium can then use it to recognize the same virus. When another virus injects the DNA, if the bacteria already has this little piece, there is a mechanism by which the bacteria recognize, "oh, this DNA that I have in the CRISPR locus is the same that is the one that is being injected." This is similar to what antibodies do in our system with antigens, with things that infect us. They have information that was generated through adaptive immunity that resulted in an antibody that then recognizes the pathogen and triggers an immune response. This little DNA of the phage is in the CRISPR system, and it is used to recognize the DNA of the phage that is invading. That then triggers an immune response that ends up with the destruction of the viral DNA that is being injected and that is how bacteria get protected.

HSF: That's fascinating. So, what specifically are you doing with this CRISPR system in order to develop the work further?

Marraffini: So, one of the aspects that we are very interested in is to understand how it works, the pure biology of it, because as I mentioned, this is a system that was discovered only maybe 10 years ago. We know only the very basics of how it works, and there are still many things that we would like to know at the mechanistic level. I mentioned that an immune reaction is triggered that destroys the virus, well, we still need to understand fully what that immune response actually is and how it destroys the virus. Another goal is to put some effort into how to repurpose these CRISPR systems to do other things that have an application. One of the applications that my lab was partly involved in, was the development of CRISPR to do genetic engineering of human cells and many other organisms. It provides a tool to do genetic manipulation of human cells much, much more easily than before.

HSF: Can you explain how that works?

Marraffini: The way that the CRISPR system kills the virus is by cutting the DNA of the virus, the genetic material. I mentioned that the CRISPR system grabs a little snippet of the DNA of the virus. DNA has a specific sequence, it is composed of four chemicals known as bases that contain information, equivalent to an alphabet of four letters; this little sequence, is composed of about 30 letters.
What the CRISPR does is that it recognizes that the same 30 letters are present in the CRISPR system and in the invading virus and then it goes and cuts the viral DNA within those 30 letters. What we and others realized is that the same ability of the CRISPR system to cut the viral DNA can be used to cut any DNA. Now the CRISPR systems have been repurposed to cut human DNA. They can be put inside human cells, and they will go and cut a specific sequence at a specific gene in the human genome. That is the technology that the CRISPR system provides for making genetic manipulations. It was known for 20 years or so that if you can cut the human genome at a specific place, the cell will need to repair the DNA, as it needs to be a linear piece of information. So 20 years ago, people realized how to fool the repair systems to make a repair with the DNA sequence that we want.

HSF: So, to pre-program how the repair goes?

Marraffini: Yes, to pre-program how the repair goes. But what was very difficult about that is that before you repair it, you need to have a cut in the DNA to be repaired. In order to cut the DNA, you need to have a tool to cut it at the desired sequence.

Recently, some proteins called sequence-specific nucleases were developed that were able to be programmed to cut a specific sequence, but that was very complicated. With CRISPR, it is very simple, because the CRISPR system is actually poised to do that. If you specify this 30 nucleotide sequence, then the CRISPR system is programmed to go and cut that in a specific place.

HSF: So, it knows how to target a place?

Marraffini: Yes, it knows how to target it, exactly where to cut it. That really made it much simpler and more efficient to make genetic manipulations of human cells and other mammalian cells, which before was much more difficult. Of course, that has a lot of ethical implications, the U.S. national academies and the European and Chinese equivalents are trying to figure out if there are going to be rules about how to use CRISPR to manipulate human cells.

Another thing we are involved in is bacterial pathogenesis. We use the same principle of using the same CRISPR system to cut whatever we want in a DNA piece, to actually cut the bacteria DNA itself and kill it. We have found that is lethal for bacteria. Once you have something that can kill bacteria, there is always the opportunity of making antimicrobials. That is one of the things we are interested in. We can now use the CRISPR system to cut the DNA of any pathogen, and in a way, we have what we call the possibility, because it is not yet a reality, of a smart anti-microbial. Most of the current anti-microbials have somewhat of a broad spectrum, so they kill many bacteria, but with the CRISPR system, we can actually kill bacteria that has a specific sequence of DNA, and not just every bacteria.

HSF: So, to kill only the bad ones?

Marraffini: Yes, now there is a lot of interest in the human microbiome, a consortia of different types of bacteria, some of them are really necessary for human health and ideally, you do not want your antibiotic to kill those, you want it to kill only the bad ones. So, with CRISPR, we may be able to do that. We published the proof of principle of that. The main problem now is how to deliver the CRISPR system, which is the reason why antibiotics were so successful, because they were just pills you take that go everywhere in the body. The CRISPR system is not a small chemical. It has at least three genetic elements that you need to deliver into bacteria and that is not very simple to do.

HSF: In the type of bacteria that you are working with now, are you targeting certain ones? I believe I read something about staphylococcus. What do you think the biggest threat may be to public health that you are trying to address?

Marraffini: Staphylococci are a big problem in the clinical setting, there are some strains that are called MRSA, methicillin-resistant staphylococcus aureus, that are resistant to multiple antibiotics. Some are resistant to all of them. Hospital acquired MRSA infections affect patients who went into surgery. However, by the late 2000s, doctors started seeing community acquired infections, and these were MRSA strains that would spread in a

community, for example, in child care centers and in sports facilities. Staphylococci live in the skin, so you just need skin contact for infection. That is what is scary, as not a lot of people go to open heart surgery, but a lot of people use day care facilities or the locker room at a sports facility. I think recently it has been less of an issue in the hospitals, as hygienic standards were improved, but community acquired infections went up. For these reasons, we are trying to use CRISPR antimicrobials against pathogenic staphylococci.

HSF: What do you think the biggest challenges are in your research field right now, either for funding or for certain types of research?

Marraffini: In general, in microbiology, I think one of the challenges is funding, I think the Hans Sigrist Prize is absolutely fantastic, because it lets us have freedom in what we do; it is very much welcome. The difficulties in funding also have an effect, not only on the current labs, but also on students, who see microbiology as a very difficult field. There are many other translational sciences, where they study more applied things, so many students tend to go into these other disciplines. Throughout the history of medicine, the study of microbiology has proven to be very important for many reasons. One is the pathogenesis aspect of microbiology, and the other aspects are the development of many of the key technologies that we have today in medicine. This CRISPR mechanism is today somewhat revolutionizing human genetics. It is being adopted by a lot of labs, and it is going to have many human applications and implications. However, when I first started, and began to do experiments on CRISPR, the people who were studying this system were like me, interested in microbiology and how bacteria defend themselves against viruses, which in principle, does not have direct relevance to anything, it is just curiosity-driven. Forty years ago, from studying the same interaction between bacteria and their viruses, scientists discovered restriction enzymes, which are also able to cut the DNA of phages, but they can also be repurposed to cut any type of DNA, which led to something called recombinant DNA technology. The discovery of antibiotics is also tightly related to people who were studying how bacteria protect themselves from other bacteria, or how fungi protect themselves. These are microbiology problems that in the end became very useful. Therefore, the restricted funding of microbiology is not a good thing.

HSF: So you feel that these foundational studies, which are not generally funded by a company with an application, are very important?

Marraffini: Absolutely, nowadays, many funding agencies are becoming stricter about what they expect research to produce, they want a goal in public health to be met specifically, but you still have to maintain basic research to come up with new things. You cannot do that strictly from application-based studies.

HSF: Can you give me your impressions of the University of Bern? How have you enjoyed your visit?

Marraffini: Today, Prof. Endimiani gave me a tour of the Institute (IFIK), and I was really impressed, because what I saw is the proximity between the clinical side and the academic research side, especially in a small setting. It seems there is a really strong fundamental connection between the research side and the hospital side, which is what you want when you are studying antibiotic resistance. You want to get the isolates that come from real infections and people. Most antibiotic resistance comes from the spreading of plasmids, and the work that Prof. Endimiani is doing focusing on plasmids is critical to solve this problem. Bacteria have these little circular pieces of DNA that can be transferred from one bacterium to another with a very high efficiency, and very quickly, a population can become resistant to a particular antibiotic. Prof. Endimiani's new ideas on how to fight this transfer are really clever.